Enhanced red blood cell production mediated by the hormone erythropoietin (Epo) is a well-known adaptive response of humans to hypoxia (Bunn & Poyton (1996) Physiological Rev. 76, 839-845). Normally produced by the adult kidney and the fetal liver, Epo stimulates the development of Epo receptor (EpoR) expressing red blood cell precursors in the bone marrow.
Epo is a glycoprotein (46 kDa) hormone produced by renal interstitial cells within the kidney that regulate the production of red blood cells in the marrow. These cells are sensitive to low arterial oxygen concentration and will release erythropoietin when oxygen is low (hypoxia). Erythropoietin stimulates the bone marrow to produce more red blood cells thereby increasing the oxygen carrying capacity of the blood. Epo exerts its effect by binding to EpoR on the surface of erythrocyte precursors in the bone marrow. Epo expression, however, has been observed on cells from other normal tissues in addition to renal interstitial cells including enterocytes, trophoblast and neuronal cells. The protein sequence for Epo, 193 amino acids in length, can be found in GenBank under Accession No. 1104303A (Jacobs et al. (1995) Nature 313, 806-810), which is incorporated herein by reference in its entirety.
The protein sequence for the erythropoietin receptor, 508 amino acids in length, can be found in GenBank, under Accession No. AAA52403 (Jones et al. (1990) Blood 76, 31-35), which is also incorporated herein by reference in its entirety.
The measurement of Epo in the bloodstream can indicate bone marrow disorders or kidney disease. Normal levels of erythropoietin are 0 to 19 mU/ml (milliunits per milliliter). Elevated levels can be seen in polycythemia rubra vera, a condition characterized by enlargement of the spleen and the increased production of red blood cells by bone marrow. Lower than normal values are seen in chronic renal failure leading to anemia. Chronic renal failure leads to anemia, in part, because of the progressive absence of adequate Epo production for the maintenance of erythropoiesis.
Hypoxia is a predominant feature of solid tumors, which comprise approximately ninety percent of all human cancers. Adaptive responses to hypoxia in solid tumors has been correlated with enhanced aggressiveness, reduced tumor cell death and diminished tumor response to both radiation and chemotherapy. Epo expression has been observed in different hematopoietic and non-hematopoietic malignancies and has been shown to mediate autonomous growth of erythrocytic leukemia cells expressing EpoR.
Epo and EpoR expression has recently been demonstrated in several other normal tissues in addition to the kidney (Marti et al. (1996) Eur. J. Neurosci. 8, 666-676; Kling et al. (1998) Pediatr. Res. 43, 216-221; Juul et al. (1999) Pediatr. Dev. Pathol. 2, 148-158; Juul et al. (1999) Pediatr. Res. 46, 263-268; Benyo & Conrad (1999) Biol. Reproduct. 60, 861-870) suggesting a wider biological role for Epo signaling. Ectopic Epo expression also has been observed in several different haematopoietic and non-haematopoietic malignancies (Mitjavila et al. (1991) J. Clin. Invest. 88, 789-797; Tachibana et al. (1991) Neurosurg. 28, 24-26) and can, in fact, mediate autonomous growth of EpoR expressing erythrocytic leukemia cells (Mitjavila et al. (1991) J. Clin. Invest. 88, 789-797). Other studies examining the effects of recombinant erythropoietin on cell lines derived from a wide range of tumors including two breast cancer cell lines determined that no significant stimulation of cancer cell proliferation occurred in response to Epo, in particular low doses of erythropoietin.
In solid neoplasias, adaptive responses to hypoxia are correlated with enhanced aggressiveness (Schmaltz et al. (1998) Mol. Cell. Biol. 18, 2845-2854), poor responses to chemo- and radiation-therapy (Kalra et al. (1993) Int. J. Cancer 54, 650-655; Brown (1999) Cancer Res. 59, 5863-5870), and reduced apoptosis (Graeber et al. (1996) Nature 379, 88-91). Many common human cancers over-express the hypoxia-inducible transcription factor HIF-1, which regulates the expression of Epo as well as several genes required for enhancing hypoxic survival of cancer cells including genes coding for glycolytic enzymes, glucose transporters, and vascular endothelial growth factor (Semenza (1999) Ann. Rev. Cell. Dev. Biol. 15, 551-578). The role of Epo and EpoR in the mechanisms by which hypoxic cancer cells gain a growth advantage and escape cell death has not been previously elucidated.
Tumor hypoxia is recognized as a major factor in tumor resistance to chemotherapy and radiation therapy although the underlying mechanisms are unknown. Hypoxia induces adaptive responses in cells largely by activating the expression of several genes under the regulation of hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of HIF-1α and HIF-1β subunits. The HIF-1α subunit is unique to HIF-1 while HIF-1β can dimerize with several different transcription factors. A decrease in cellular oxygen concentration increases HIF-1α protein levels, which in turn determines the level of HIF-1 activity. Hypoxic regions of gliomas have been shown to highly express HIF-1α (Zagzag et al. (2000) Cancer 88, 2606-2618) as well as several HIF-1 regulated genes including vascular endothelial growth factor and glucose transporters.
Epo is among the most-studied HIF-1-regulated genes. This glycoprotein hormone, normally produced by the kidney and fetal liver, acts via EpoR to stimulate growth, prevent apoptosis, and induce differentiation of red blood cell precursors. Epo and EpoR are also expressed in the nervous system during development and in adult brain following ischemic injury (Sasaki et al. (2001) News Physiol. Sci. 16, 110-113). Several in vitro and in vivo studies have demonstrated a potent neuroprotective effect of erythropoietin in cerebral ischemia, which appears to be mediated by an inhibition of apoptosis (Cerami (2001) Semin. Hematol. 38, 33-39; Digicaylioglu et al. (2001) Nature 412, 641-647). EpoR activation stimulates tyrosine phosphorylation of several proteins, which impact upon multiple signaling pathways capable of promoting cell survival (Cheung et al. (2001) Nephron 87, 215-222). These include the activation of Jak-2, STAT-5, and the PI3K pathway.
Recombinant Epo is now being used therapeutically in cancer patients. Specifically, Epo is used to treat anemia in cancer patients undergoing chemotherapy. Chemotherapy-induced anemia is mainly due to the renal impairment induced by anti-neoplastic drugs which leads to insufficient renal production of Epo with a consequent reduction in red blood cell production. Epo therapy has therefore been widely used as a substitute for blood transfusions in patients undergoing chemotherapy, particularly in breast cancer patients. Accordingly, the role of Epo in the development and progression of certain solid tumors needs to be elucidated.